Field portable analysis of biological agents depends on the ability to prepare unknown samples for analysis. A major component of sample preparation is lysing and solubilizing biological agents. Sample preparation is complicated by the wide variability in the stability of biological agents to the effects of lysis and solubilization. A variety of techniques have been developed for lysing viruses and bacterial agents, examples of which include chemical and detergent lysis, enzyme treatment, sonication, heating, and glass bead milling. Bacterial spores, for example, are extremely resistant to lysis and solubilization, often requiring a combination of the aforementioned techniques. However, many of these lysis techniques complicate analysis due to the addition of chemical additives or proteins to the samples which interfere with the amplification, labeling or analytical analysis.
Several sample processing techniques have been developed for the use and integration of microfluidic devices. These techniques and microfluidic devices include integrated detergent mediated lysis, laser mediated cell lysing, and electric field mediated lysis. In addition, highly integrated systems have been developed that allow for the lysis, concentration, purification, and analysis of DNA from E. coli. Many of these studies perform sample processing on relatively labile eukaryotic cell types, and bacteria. Few studies have been directed to rapidly lysing and analyzing bacterial spores for microfluidic analysis. Belgrader, et al., (“A minisonicator to rapidly disrupt bacterial spores for DNA analysis,”Analytical Chemistry, 71(19), 1999, 4232-6) previously demonstrated that sonication of Bacillus subtilis spores in the presence of silica beads was an effective technique for lysis prior to polymerase chain reaction (“PCR”) analysis of spore DNA. Taylor, et al., (“Lysing bacterial spores by sonication through a flexible interface in a microfluidic system,” Analytical Chemistry, 73(3), 2001, 492-6) followed up these studies by demonstrating real-time PCR and protein signature analysis using a similar device.
Sample preparation is complicated by the wide variability in the stability of biological agents to the effects of lysis and solubilization. Numerous techniques have been developed for lysing spores, viruses, and cells. Common examples include enzyme digestion, chemical reducing agents, sonication, heating, and glass bead milling. Bacterial spores are extremely resistant to lysing and require harsh conditions to solubilize. A common protocol is to use a reducing agent such as TCEP (tris(2-carboxyethyl)phosphine) or DTT (dithiothreitol). In order to further analyze the solubilized proteins, the reducing agent must be removed, a time-consuming and labor intensive step.
Thus, there is a continuing need to develop sample processing techniques useful for a variety of analytical techniques including protein fingerprinting and PCR. There is also a need to develop sample processing techniques useful for immunological based reagent detection and other electrophoretic analysis. In addition, there is a need to develop protocols to process extremely robust sample agents such as bacterial spores for direct integrated analysis using microfluidic devices for protein fingerprinting. There is also a need to develop devices that rapidly lyse bacterial spores for use in DNA analysis using, such as PCR, using microfluidic devices and chips.